Skeletal muscles are not all created equal. Muscle precursor cells or myoblasts are not a homogeneous population with a preordained fate, but rather represent distinct lineages derived from a variety of embryonic sources. Extrinsic factors interact with lineage in sculpting the molecular/cellular/structural traits of developing myofibers. The extraocular muscles (EOMs) are particularly unique among skeletal muscles. Our central hypothesis is that development of the novel EOM phenotype relies upon cell autonomous and non-cell autonomous regulatory mechanisms that are both shared with and strikingly divergent from those determining myoblast fates in other skeletal muscles. Specifically, we propose that neural interactions unique to the orbital environment and signaling cascades controlled by tissue-specific transcription factors direct EOM expression of a diverse array of EOM-specific, skeletal, and cardiac muscle traits that adapt it to the wide dynamic physiologic range required by eye movement control systems. A central problem is that the wealth of data from 'traditional' muscle studies is insufficient to explain the patterning and specification of distinctive EOM properties. Our recent expression profiling studies and development of an EOM cell line have provided the knowledge and tools to now identify and directly test EOM regulatory mechanisms. Specific Aim 1 will address cell autonomous regulatory mechanisms in EOM development, using immortialized EOM and hindlimb muscle cell lines in conjunction with gene/protein expression profiling. Specific Aim 2 will examine the role of the transcription factor, Pitx2, in regulating early and late features of EOM development. An allelic series of Pitx2 mutant mice will be used to determine dose dependence in regulation of both general and fiber type-specific traits. Specific Aim 3 will determine the role of a non-cell autonomous mechanism, unique to the orbital environment, in shaping EOM development. Our finding that EOM survival in organotypic co-culture is dependent upon oculomotor motoneuron innervation will be extended by assessing molecular specialization of EOM in culture with correct vs. incorrect motoneurons. The health relatedness of this proposal is that there is a clear need to understand the molecular specification of the EOM phenotype in order to comprehend both its unique functional features and differential responsiveness to metabolic and neuromuscular disease.